High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability.

A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri(rPAC(P.rett))of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEP4 gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P.pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability >3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of beta-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process.

Construction of Saccharomyces cerevisiae strain FAV20 useful in detection of immunosuppressants produced by soil actinomycetes.

The screening of microbial natural products continues to represent an important route to the discovery of novel chemicals for development of new therapeutic agents. The aim of this work was to develop an efficient method for the detection of immunosuppressive compounds produced by soil actinomycetes. Mutant strain of Saccharomyces cerevisiae, named FAV20, sensitive to FK506 was constructed by disrupting VMA22 gene using the selectable marker kanMX4 which allowed detection of integration events. Actinomycetes were isolated from different soil samples and in a newly developed test with S. cerevisiae FAV20, six strains have been identified that produce bioactive compounds with the same mechanism of action as FK506. S. cerevisiae FAV20 can be easily used as a test strain in drug screening programs based on inhibition of the calcineurin phosphatase dependent signaling pathway in the cell.

Expression and characterization of human interferon-beta1 in the methylotrophic yeast Pichia pastoris.

We describe the heterologous expression of a human interferon-beta1 in the methylotrophic yeast Pichia pastoris. Biologically active recombinant human interferon-beta1 (rHuIFN-beta1) was secreted from shake-flask-grown P. pastoris cells into the medium using the Saccharomyces cerevisiae alpha-mating factor prepro-leader sequence at the level of (1-3) x 10(5) i.u. (international units)/ml (6-12 mg/litre). An rHuIFN-beta1 with an N-terminal sequence identical with that of native HuIFN-beta1 was purified and the specific activity was determined (2-3 x 10(7) i.u./mg). It was found that the secreted recombinant protein was partially N-glycosylated.

High-level secretory expression of penicillin amidase from Providencia rettgeri in Saccharomyces cerevisiae: purification and characterization.

Heterologous production of the heterodimeric penicillin G amidase (PAC) from Providencia rettgeri was optimized in Saccharomyces cerevisiae. Several factors, including the effect of different growth and induction conditions, were identified to be critical for the enzyme overproduction and secretion. The PAC yield was significantly increased by more than 500-fold compared to that obtained in the native bacterium, and the recombinant enzyme was almost entirely secreted. Electrophoretic characterization of the secreted rPAC(Pr), which was purified over 20-fold by a combination of hydrophobic interaction and ion-exchange chromatography, demonstrated a microheterogeneity of the recombinant enzyme. The recombinant PAC(Pr) was further characterized in terms of specific activity, pH, and temperature profiles and kinetic parameters. The data presented here suggest that by overexpressing rPAC(Pr) in S.cerevisiae and purifying secreted enzyme from culture medium one can readily obtain a large amount of an alternative source of penicillin amidase with properties comparable to that of todays main industrial source of enzyme.

Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris.

Penicillin G amidase from Providencia rettgeri is a heterodimer of 92 kDa. We have previously expressed the Pr. rettgeri pac gene coding for this enzyme in Saccharomyces cerevisiae, and now we report the expression and characterization in the methylotrophic yeast Pichia pastoris. The recombinant catalytically active enzyme (rPAC(Pr)) was secreted from shake flask-grown P. pastoris cells into the medium at a level of approximately 0.18 U ml(-1). This yield of rPAC(Pr) was higher, by two orders of magnitude, than that obtained using a single-copy expression plasmid in S. cerevisiae. In addition, the secreted recombinant enzyme was entirely N-glycosylated. The recombinant PAC(Pr) was further characterized in terms of specific activity, kinetic parameters and thermostability. Except the significantly higher thermostability of the glycosylated rPAC(Pr) produced in P. pastoris, the other parameters were very similar to those of the corresponding non-glycosylated enzymes produced in bacteria or in S. cerevisiae. The higher thermostability of this recombinant enzyme has a clear industrial advantage.

The acetyl xylan esterase of Bacillus pumilus belongs to a family of esterases with broad substrate specificity.

The Bacillus pumilus gene encoding acetyl xylan esterase (axe) was identified and characterized. The axe gene was expressed and the recombinant enzyme produced in Escherichia coli was purified and characterized. The recombinant enzyme displayed similar properties to the acetyl xylan esterase (AXE) purified from B. pumilus. The AXE primary structure was 76% identical to the cephalosporin C deacetylase of B. subtilis, and 40% to two recently identified AXEs from Thermoanaerobacterium and Thermotoga maritima. These four proteins are of similar size and represent a new family of esterases having a broad substrate specificity. The recombinant AXE was demonstrated to have activity on several acetylated substrates, including on cephalosporin C.